Stubomycin Was Isolated from the Culture Broth and Mycelia of Streptomyces Strain No. KG-2245 by UMEZAWA Et Al. in 1981.1) Stubo

VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 301

STUDIES ON THE BIOLOGICAL ACTIVITY OF STUBOMYCIN

KANKI KOMIYAMA,KEN-ICHi EDANAMI, AKIRA TANOH, HIROSHI YAMAMOTOand IWAO UMEZAWA

The Kitasato Institute, Minato-ku, Tokyo 108, Japan

(Received for publication November 30, 1982)

Stubomycin showed direct cytotoxic activity on mammalian cells, yeast, and fungi, and rapid hemolytic activity on mouse erythrocytes. The rate and extent of the cytotoxic and hemolytic activities decreased at lower temperatures. Studies with radioactive precursors revealed that a marginal cytocidal concentration of the antibiotic inhibited synthesis of DNA, RNA, and protein of leukemic cells at almost the same rate. Stubomycin did not show any mutagenicity on mammalian cells and bacteria i.e. the induction of revertants on six bacterial strains, and chromosomal aberrations, sister chromatid exchanges, and the induction of cells resistant to 6-thioguanine on Chinese hamster cells (DON D-6). The antagonistic effect of various kinds of lipids including phospholipids, cholesterol, olive oil and squalene was studied. Significant antagonism of stubomycin against anti-Saccharomyces cerevisiae activity was observed with phospholipids except for egg lecithin and with cholesterol. The primary action of the antibiotic seems to be to change the cell surface and ultimately the lysis and death of the cells.

Stubomycin was isolated from the culture broth and mycelia of Streptomyces strain No. KG-2245 by UMEZAWAet al. in 1981.1) Stubomycin has been shown to have marked in vivo antitumor activity on Ehrlich ascites carcinoma, P 388 leukemia, Meth A fibrosarcoma, etc. The antibiotic is also active against Gram-positive bacteria, some fungi, and HeLa cells in vitro.') Recently, OMURAet al. have eluci- dated the structure of stubomycin as a macrocyclic lactam involving (3-phenylalanine.3) This report describes preliminary studies on the cytocidal activity of stubomycin mainly using mammalian cells, yeast, and fungi.

Materials and Methods

Chemicals N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG, Nakarai Chemicals Ltd., Kyoto), 2-nitrofluorene .(Aldrich Chemical Co ., USA), and 9-aminoacridine (Tokyo Kasei Kogyo Co., Tokyo) were dissolved in dimethyl sulfoxide. Ethyl methanesulfone and 6-thioguanine (EMS and 6-TG, Sigma Chemical, USA), mitomycin C (Kyowa Hakko Kogyo Co., Tokyo), and benzylpenicillin (Toyo Jozo Co., Shizuoka) were dissolved in phosphate buffer or water. [3H]Thymidine ([3H]TdR, 16.2 Ci/mmole), [3H]uridine ([3H]UR, 38.3 Ci/mmole), and [3H]leucine (158.0 Ci/mmole) were obtained from the Radiochemical Center, Amersham, UK. Cardiolipin, phosphatidylserine, and egg lecithin (Kitasato Institute, Tokyo), olive oil and cholesterol (Wako Pure Chemi- cal Industries, Ltd., Osaka), and squalene (Kuraray Co., Osaka) were dissolved in ether or methanol. Cells and Media Salmonella typhimurium strain numbers TA98, 100, 1535, 1537, and 1538, and Escherichia coli WP 2uvrA were provided by Dr. T. KADA,National Institute of Genetics. DON D-6 cells, derived from Chinese hamster lung cells, were maintained in EAGLE'Sminimum essential medium (MEM) supplemented with fetal calf serum (10 %), sodium pyruvate (1 MM), L-serine (0.2 mm), penicillin (100 u/mI), and streptomycin (100,ag/ml) at 37°C in an atmosphere of 5 % CO, in air. Mouse leukemic EL-4 cells 302 THE JOURNAL OF ANTIBIOTICS MAR. 1983

were maintained in MEM supplemented with 10 % calf serum, and cells in the logarithmic growth phase were used for the experiment. Saccharomyces cerevisiae and Pyricularia oryzae have been maintained in our laboratory on potato agar and rice straw agar respectively. To determine the effect of stubomycin on these microorganisms, two media were used: (1) 0.3 % yeast extract, 0.3 % peptone, 3 % glucose, and 0.3 % Trypticase soy broth for S. cerevisiae; and (2) 0.5 % peptone, 0.3 % yeast extract, and 3 % glucose-potato extract solution (200g of potato was boiled in 1 liter of water for 30 minutes, and the solution was used as potato extract) for P. oryzae. Measurement of Cytotoxicity To determine the cytotoxicity of stubomycin on mammalian cells, EL-4 cells (2 x 106) in 2 ml of medium were placed in 30-mm Petri dishes and incubated for 48 hours. Then, 0.01 ml of different concentrations of stubomycin solution were added to each culture dish, and thereafter, the number of trypan blue excluded cells was counted periodically using a hemocytometer. In case of antibacterial activity, the antibiotic was added to suspensions of S. cerevisiae (optical density: 0.092) and P. oryzae (optical density: 0.043) in 4 ml of medium, and the mixtures were shaken at 27°C. The growth of S. cerevisiae was followed by determining the absorbancy of cultures at 660 nm. In case of P. oryzae, the culture was gently dispersed by a homogenizer (Biotron), and the growth of P. oryzae was followed by determining the absorbancy of cultures at 660 nm. Values are the mean of three samples. Measurement of Viability EL-4 cells (7 x 105) in 7 ml of MEM were exposed to 0.05 ml of different concentrations of stubomycin solution for 5 to 60 minutes. Then at the end of each incubation time, cells were rinsed three times with fresh MEM, and were suspended in 6 ml of culture media. Two ml of each cell suspension were plated on 30-mm Petri dishes. Viable cells were counted after incubation at 37°C for 72 hours. For viable count determination on S. cerevisiae, the microorganism suspended in medium was exposed to different concentrations of stubomycin for 10, 30, or 60 minutes. After incubation, the cells were washed three times with 5 ml of ice-cold medium by centrifugation, diluted with the medium, and plated on potato dextrose agar. After 48 hours of incubation at 37°C, the colonies were counted. Effect of Temperature on Anti-S. cerevisiae Activity of Stubomycin S. cerevisiae (optical density: 0.24) was exposed to 1.25 ,ug/ml of stubomycin for 30 minutes at different temperatures. At the end of incubation, the cells were centrifuged at 0°C (600 x g, 2 minutes). The cells were then resuspended in the growth medium. After incubation for 6.5 hours at 27°C, the absorbancy of the cultures was determined at 660 nm. Measurement of Macromolecular Synthesis EL-4 cells (1 X 106 in 2 ml of MEM) were mixed with stubomycin and various precursors, and incubated at 37°C for 60 minutes. The cells were then collected on a Millipore filter and rinsed 3 times with ice-cold 5 % trichloroacetic acid. The radioactivity of acid-precipitable material on the filter was determined by a Packard Tri-Carb liquid scintillation spectrometer. Mutagenicity of Stubomycin Effect of Stubomycin on Induction of Revertants in Six Bacterial Strains: The method for detec- tion of mutagenicity as described by AMESet al.') and YAHAGiet al.5>was used. Briefly, stubomycin was dissolved in DMSO and diluted with sterilized distilled water. The antibiotic solution (0.1 ml) was mixed with or without 0.5 ml of S-9 solution, and these mixtures were combined with 0.1 ml of bacterial suspension (1 x 109), then incubated at 37°C for 20 minutes. After incubation, 2 ml of soft agar were added to the mixture and the mixture was plated on 90-mm Petri dishes. After 2 days of incubation at 37°C, the number of revertant colonies were counted. As positive mutagen controls, 2-aminoanthracene for the metabolic study and ENNG, 2-nitrofluorene, and 9-aminoanthracene for the non-metabolic study were used according to the bacteria applied as test organism. DNA Damaging Effect of Stubomycin Determined by the rec-Assay: The rec-assay with Bacillus subtilis was performed by the method described by KADAet al.°'7> Briefly, strains of M 45 or H 17 were VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 303 cultured overnight in brain heart infusion broth. Each culture was streaked radially on the surface of broth agar, and a paper disk (diameter: 8 mm) containing the drug test solution was placed over the starting point of the streaks. The plates were kept at 4°C for 30 minutes, then incubated overnight at 37°C. The lengths of the inhibitory zones were measured. Effect of Stubomycin on Chromosomal Aberrations and Sister-chromatid Exchanges in DON Cells : DON cells (1 x 101) in 5 ml of medium were plated on 60-mm Petri dishes and incubated at 37°C for 15 hours. After plating, the cells were exposed to stubomycin or EMS for 31 - 50 hours in medium with or without 5'-bromodeoxyuridine (10-1 M). Three hours prior to harvest, Colcemide was added to give a final concentration in the culture of 0.05 µg/ml. At the time of harvest, the trypsinized cells were gently centrifuged, resuspended in hypotonic saline (0.075 M KCI) and allowed to stand for 10 minutes. After fixation with a mixture of methanol - acetic acid (3: 1), the cell suspensions were dropped onto slides, allowed to air dry and stained with Giemsa. Effect of Stubomycin on Induction of Mutation Resistant to 6-TG in DON Cells: DON cells (2 x 101) in 4.5 ml of medium were plated in 25-cm' plastic flasks (Corning, USA). After plating, 0.5 ml of the antibiotic or EMS solution was added to the culture which was then reincubated for 15.5 hours. The resulting culture was washed with phosphate buffered saline, resuspended in fresh MEM, and further incubated for 8.5 hours (day 0). To express mutation, the cells were subcultivated every other day, and the cells were trypsinized to select the mutants on days 13 or 15. The resulting cells (5 x 104) in 10 ml of medium containing 6-TG (10-1 M) were plated on 100-mm Petri dishes. In addition, 2 x 101 intact DON cells in 5 ml of medium were plated on 60-mm Petri dishes to determine the viability of the cells. After cultivation of drug exposed and intact cells for 8 days, the colonies formed on Petri dishes were fixed, stained, and counted. Measurement of Hemolysis Red blood cells were obtained from normal adult mice (strain ddY). After being washed three times in physiological saline, the cell suspension was diluted with saline to produce an absorbancy of about 1.0 at 550 nm when complete hemolysis occurred in water. The mixtures of erythrocytes and different concentrations of stubomycin were incubated at 37°C for 60 minutes and then centrifuged (1,000 rpm for 5 minutes). The absorbancy of supernatants was measured at 550 nm, and the percent hemolysis was calculated as follows:

Percent hemolysis = AbAbsorbance of hemolysis by stubomycin - x 100 sorbance of complete hemolysis by water Effect of Calf Serum on Cytotoxic Activity of Stubomycin EL-4 cells (1 x 101 in 2 ml MEM) were suspended in MEM supplemented with different concentrations of calf serum. Then, stubomycin (0.2 Mg/ml) was added to the cell suspensions. After this mixture was incubated for 30 minutes at 37°C, the cells were washed three times with MEM and resuspended in MEM supplemented with 10%. calf serum. The cells were counted after 4 days of incubation. Effect of Lipids on Antimicrobial Activity of Stubomycin Four cultures of S. cerevisiae were mixed with 0.01 ml of lipid and stubomycin solutions to produce final concentrations of 10 µg/ml and 0.63 Fig/ml respectively. After incubation at 27°C for 7 hours, the cultures were washed three times with a methanol - ether (1: 1) solution by centrifugation to remove the lipids. The resulting precipitate was resuspended in saline, and the absorbancy of the suspension was determined at 660 nm.

Results

Growth Inhibitory Effect of Stubomycin on Various Cells Cells growing in the logarithmic phase in vitro were mixed with different concentrations of stubomycin, and their growth was observed periodically. As shown in Figs. 1 - 3, the antibiotic remarkably inhibited the growth of EL-4 cells, P. oryzae and S. cerevisiae. 304 THE JOURNAL OF ANTIBIOTICS MAR. 1983

Fig. 1. Effect of stubomycin on the growth of EL-4 Fig. 3. Effect of stubomycin on the growth of leukemic cells. S. cerevisiae.

Incubation time (hours)

Incubation time (hours) Fig. 4. Effect of stubomycin on the viability of S. cerevisiae.

Fig. 2. Effect of stubomycin on the growth of P. oryzae.

Incubation time (hours) Incubation tine (minutes)

Next, to examine cytocidal activities of stubomycin on EL-4 cells and S. cerevisiae, the cells were mixed with stubomycin and incubated for 10, 30, or 60 minutes. At the end of each incubation time the cells were washed and incubated again to determine their growth. As shown in Figs. 4 and 5, remarkable cytocidal activity was observed on S. cerevisiae and EL-4 cells at concentrations of 2.5 and 0.8 ,gag/ ml respectively.

Effect of Temperature on the Activity of Stubomycin The effect of temperature on the growth inhibitory effect of stubomycin on cells was examined. When S. cerevisiae was incubated with 1.25 pg/ml of stubomycin at 0 or 10°C for 30 minutes, no growth VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 305

Fig. 5. Effect of stubomycin on the viability of EL- Fig. 6. Effect of temperature on the anti-S. cerevisiae 4 leukemic cells. activity of stubomycin.

Control

Control 0.05jiq/ml

0.2Ug/ml

1 .25 pg/ml

0.&pg/ml

Temperature (°C)

Fig. 7. Effect of stubomycin on the synthesis of ma- cromolecules in EL-4 cells. Incubation time (minutes)

inhibition was observed. However, cells treated with stubomycin at 27°C or 37°C for 30 minutes [3H]Leucine suffered a considerable loss in growth (Fig. 6). [3H]UR Effect of Stubomycin on Macromolecular

Synthesis in EL-4 Cells [3H]TdR To determine the effect of stubomycin on the synthesis of DNA, RNA and protein, the uptake Drug concentration (pg/ml) of various precursors into EL-4 cells was observed. As shown in Fig. 7, when cells were exposed to various concentrations of stubomycin for 60 minutes, the uptakes of [3H]TdR, [3H]UR, and [3H]leucine were equally inhibited at all drug concentrations. Mutagenicity of Stubomycin The effects of stubomycin on induction of revertants on six bacterial strains were studied. There were different sensitivities to stubomycin among bacterial strains, but no increases in the number of revertant colonies were observed in any of the strains. In contrast, positive control agents such as ENNG increased the number of revertant colonies (Table 1). The DNA damaging effect of stubomycin was determed by the rec-assay method. As shown in Table 2, there was no difference in growth inhibitory zones between strains of B. subtilis M 45 and H 17. In contrast, mitomycin C used as positive control showed a remarkable inhibition of the rec- strain. The effects of stubomycin on chromosomal aberrations, sister-chromatid exchanges, and the induction of cells resistant to 6-TG were studied using Chinese hamster cells (DON D-6). The antibiotic slightly increased numerical aberrations of chromosomes at a concentration of 0.24Itg/ml, but no increases in structural aberrations and rate of sister-chromatid exchanges were observed (Table 3). Almost no increase in the induction of mutation resistant to 6-TG in Chinese hamster cells was observed even at a nearly lethal dose of the antibiotic, whereas EMS used as positive control induced 30 - 40 fold more mutations than the vehicle control (Table 4). 306 THE JOURNAL OF ANTIBIOTICS MAR. 1983

Table 1. Effect of stubomycin on induction of revertants in six bacterial strains,

Number of revertants/plate With or Concentrations without Agents Base change type Frame shift type (µg/plate) S-9 mixture TA100 TA1535 WP 2uvrA TA98 TA1537 TA1538

Vehicle control 283 63 48 51 13 33 Stubomycin 5,000 57 25 30 1,000 54 33 42 500 61 42 48 100 1 61 32 39 0 1 50 60 61 46 31 0 2 10 37 70 33 44 7 14 5 88 15 15 1 204 11 25 0.5 173 16 28 Vehicle control + 266 18 114 109 26 61 Stubomycin 5,000 12 38 63 1,000 + 18 41 79 500 + 14 51 81 100 + 0 19 37 92 0 4 50 + 9 22 60 74 0 18 10 + 95 17 51 61 22 39 5 + 107 26 50 1 + 218 31 62 0.5 + 247 28 50 Positive control ENNG** 10 4,712 5 2,124 2 680 2-Nitrofluorene 5 874 2 149 9-Aminoacridine 10 33 2-Aminoanthracene 40 366 271 1 61 85 0.5 + 528 300

* : Not tested . **: N-Ethyl-N'-nitro-N-nitrosoguanidine .

Table 2. Mutagenicity of stubomycin determined by Fig. 8. Hemolytic activity of stubomycin on mouse rec-assay. red blood cells. Mouse red blood cells were mixed with the indi- Concen- Inhibitory cated amounts of antibiotics, and incubated at 37°C tration zone (mm) Difference (mm) for 1 hour. (µg/disc) M 45 H 17 Stubomycin Vehicle control 0 0 0 0 Stubomycin 500 4.7 5.4 -0 .7 100 4.7 4.4 0.3 50 5.7 5.0 0.7 10 4.0 4.3 -0 .3 Amphotericin B 5 4.0 3.7 0.3 1 2.0 1.7 0.3 0.5 2.0 2.0 0 Dose (u9/m1) Negative control 13.0 13.7 -0 .7 Benzylpenicillin 100 Positive control 12.0 5.0 7.0 Mitomycin C 7 VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 307

Table 3. Effect of stubomycin on chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells.

Chromosomal aberrations/cell

Concentrations Mitotic Structural aberrations SCEs*/cell Agents index Numerical (mean± (M) (per hour) Chromatid Chromosome Others aber- S.D.) rations Gaps Breaks Gaps Breaks

Vehicle control 3.02 0.04 0 02 0 0 0.02 0.10 4.54±1.99 Stubomycin 5 x10-7 0.12 0.04 0.02 0 0 0.06 0.28 ND 2.5 x 10-7 2.95 0.04 0 0 0 0.06 0.08 4.64±2.01 1.3x10-7 3.07 0.02 0 0 0 0.06 0.08 4.92±1.70 Ethyl methane- sulfonate 10-3 2.42 0.06 0.06 0.08 0.02 0.02 0.10 25.74±6.64

* Sister-chromatid exchanges .

Table 4. Effect of stubomycin on induction of mutation resistant to 6-thioguanine in Chinese hamster cells.

Mutation Treatment Expression Cell survival Number of mutants frequencies (M) time (days) (%) per dish (mutants/105 survivors)

Vehicle control 13 98.9±2.7 0.44±0.73 0.89 15 86.5 ±5.3 0.33±0.41 0.76 Stubomycin 5 x 10-7 15 92.0±6.9 0 0 2.5 x 10-7 13 110.7±5.1 0.89±1.27 1.61 15 90.4±6.2 1.50±1.64 3.32 1.3x10-7 13 97.1 ±2.5 0.80±0.92 1.65 15 97.3±7.1 0.66±0.52 1.36 EMS 2 x 10-3 13 95.9±3.8 12.50±5.45 26.08 15 75.9±5.2 11.00±1.52 28.99

Fig. 9. Effect of incubation time on hemolytic acti- Fig. 10. Effect of temperature on hemolytic activity vity of stubomycin. of stubomycin. Mouse red blood cells were mixed with the indi- Mouse red blood cells were mixed with a con- cated amounts of stubomycin, and incubated at 37°C. centration of I pg/ml of stubomycin, and incubated The hemolysis was observed periodically. at different temperatures.

42°C

37°C

22°C 5 p g/ml I Ug/ml

0.2 ug/ml

0°C

Control

Incubation tine (minutes) Incubation time (minutes) 308 THE JOURNAL OF ANTIBIOTICS MAR. 1983

Hemolytic Activity of Stubomycin on Mouse Erythrocytes A constant number of mouse erythrocytes was incubated with varying amounts of stubomycin at 37°C for 1 hour. As shown in Fig. 8, stubomycin induced almost complete hemolysis at a concentration of 0.3 yg/ml. Amphotericin Bused as positive control also induced significant hemolysis. Next, the hemolytic activity was determined periodically at different concentrations of the antibiotic, and the results are shown in Fig. 9. Rapid hemolysis was observed within 5 minutes at 5 tag/ml, and this hemolysis at each dose reached a maximum after 20 - 30 minutes. The effect of temperature on hemolytic activity of stubomycin was examined periodically. When mouse erythrocytes were incubated with 1 ,eeg/ ml of the antibiotic at 0°C, no marked hemolysis occurred. However, remarkable hemolysis was noted as the temperature increased (Fig. 10).

Effect of Calf Serum on Cytocidal Activity To determine the effect of calf serum on cytocidal activity of the antibiotic against EL-4 cells, the cells were suspended in MEM supplemented with various concentrations of calf serum and 0.2 tg/ml of stubomycin for 30 minutes at 37°C. As shown in Fig. 11, stubomycin applied to EL-4 cells in the medium not containing any serum markedly inhibited their growth at a concentration of 0.2 fig/mi. In contrast, the anti-EL-4 cell activity of the antibiotic tended to decrease with increasing amounts of serum.

Effect of Lipids on Antibacterial Activity of Stubomycin The effect of lipids on the antibacterial activity of stubomycin was studied using S. cerevisiae. The activity was significantly reduced by phosphatidylserine, olive oil, and cardiolipin. However, egg lecithin and cholesterol did not reduce the activity of stubomycin (Fig. 12).

Fig. 12. Effect of several lipids on anti-S. cerevisiae Fig. 11. Effect of calf serum on the anti-EL-4 acti- activity of stubomycin. vity of stubomycin. S. cerevisiae were incubated with lipid or lipid EL-4 cells were incubated with (•) or without (0) and stubomycin. Control culture was added only stubomycin in the medium supplemented with vari- with the antibiotic. Values are the mean of 3 ous concentration of calf serum. samples.

Phosphatidyl- serine Cardiolipin

Olive oil

Squalene

Cholesterol

Lecithin

Control

Concentration of calf serum (v/v %) Absorbance at 660 nm (O with lipid, =with lipid and stubomycin)

Discussion

The antitumor antibiotic, stubomycin, possesses marked cytotoxic activity against mammalian cells, yeast, and fungi in vitro. Although the cells were exposed only once to stubomycin for a short VOL. XXXVI NO. 3 THE JOURNAL OF ANTIBIOTICS 304 period of time, cell growth was inhibited. The hemolytic activity of stubomycin on mouse erythrocytes was also rapid. These results suggest that stubomycin is quickly incorporated into the cells. The biological activity of the antibiotic described above depended on various conditions, i.e. marked activities. were observed at higher temperatures than at lower ones, and were inhibited by phosphatidylserine, cardiolipin, cholesterol, etc. It is well known that polyene antibiotics mediate a change in the cellular permeability of a number of organisms, thus promoting leakage of important cellular constituents.8~12) Previously, it has been de- monstrated by many researchers13-17) that exogenously added sterols can protect sensitive organisms from the action of polyenes, and polyene antibiotics induce hemolysis of rat and human erythrocytes.18~22) Therefore, it is suggested that the biological activities of stubomycin are similar to those of polyenes. The effects of stubomycin on hemolysis are similar to those of filipin i.e. rapid hemolysis induced at lower concentrations (1-2 tg/ml) without at time lag for 10-15 minutes.22) Furthermore, the drug concentration of stubomycin that causes cell death and hemolysis is almost the same, and a similar phenomenon was observed in a study of polyene antibiotics.21) While the antimicrobial activity of polyene antibiotics is generally reduced by sterols, that of stubomycin was not reduced by cholesterol. On the other hand, the antimicrobial activities of macrocyclic lactam antibiotics azalomycin F24.25)and copiamycin,26•27)were more strongly reduced by phospholipids with unsaturated fatty acid and basic hydrophilic groups, and were moderately reduced by egg lecithin.28) In the present study, some phospholipids reduced the anti-Saccharomyces cerevisiae activity of stubomycin but there was no reduction by egg lecithin. Stubomycin is also a macrocyclic lactam antibiotic although the mechanism of action seems to differ from that of copiamycin and azalomycin F. The anti-EL-4 cell activity of the antibiotic tended to decrease with increasing amounts of calf serum. Because calf serum contains different and relatively large amounts of lipid,29) it is considered that these lipids inhibited the cytocidal activity of stubomycin. Most antitumor antibiotics with direct cytotoxic activity used clinically at present, primarily inhibit DNA synthesis,30) and most of them cause mutation.'4,31) Stubomycin showed DNA, RNA, and protein synthesis inhibition at almost the same time and to the same degree. On the other hand, stubomycin did not show any mutagenesis in mammalian cells and bacteria. The main mechanism of mutation is considered to be DNA damage and errors in replication of DNA. From the data obtained in the present experiment, it seems that stubomycin can mediate a change in the cell surface ultimately leading to lysis and death of the cells.

Stubonrycin

copianycin Azalomycin F4a 310 THE JOURNAL OF ANTIBIOTICS MAR. 1983

Acknowledgment

The authors are indebted to Mr. A. SoNO for his collaboration with the mutagenic studies. We are grateful to Dr. T. HATA for his kind advice. This experiment was supported by Grants in Aid for Cancer Research from the Ministry of Education, Culture and Science in Japan.

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